Peptides derived from pro-ACTH/endorphin will be purified from spent AtT-20 tumor cell culture medium by ion exchange chromatography, isoelectric focusing, and gel filtration. Purified 16K fragment, beta-lipotropin, gamma-lipotropin, glycosylated ACTH and pro-ACTH/endorphin will be subjected to structural studies and sequence analysis and used to develop immunoassays and to carry out bioassays. Subcellular fractions (e.g. secretory granules, rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi vesicles) will be prepared from mouse tumor cells, rat anterior pituitary and rat intermediate pituitary. These subcellular organelles will be used for in vitro studies of the post-translational processing of pro-ACTH/endorphin-derived peptides, in particular, the ability of the various fractions to carry out acetylation, amidation, glycosylation and proteolytic cleavage will be studied. The biosynthesis and structure of peptides related to ACTH, beta LPH and 16K fragment in rat hypothalamic neurons will be studied by immunoassay, in vitro incubations of isolated cells with labeled precursors, and immunoprecipitation. Studies of opiate and melanotropic peptides in lower vertebrates will be initiated.